Efficient hydroxylation of flavonoids by using whole-cell P450 sca-2 biocatalyst in Escherichia coli

The hydroxylation is an important way to generate the functionalized derivatives of flavonoids. However, the efficient hydroxylation of flavonoids by bacterial P450 enzymes is rarely reported. Here, a bacterial P450 sca-2mut whole-cell biocatalyst with an outstanding 3′-hydroxylation activity for the efficient hydroxylation of a variety of flavonoids was first reported. The whole-cell activity of sca-2mut was enhanced using a novel combination of flavodoxin Fld and flavodoxin reductase Fpr from Escherichia coli. In addition, the double mutant of sca-2mut (R88A/S96A) exhibited an improved hydroxylation performance for flavonoids through the enzymatic engineering. Moreover, the whole-cell activity of sca-2mut (R88A/S96A) was further enhanced by the optimization of whole-cell biocatalytic conditions. Finally, eriodictyol, dihydroquercetin, luteolin, and 7,3′,4′-trihydroxyisoflavone, as examples of flavanone, flavanonol, flavone, and isoflavone, were produced by whole-cell biocatalysis using naringenin, dihydrokaempferol, apigenin, and daidzein as the substrates, with the conversion yield of 77%, 66%, 32%, and 75%, respectively. The strategy used in this study provided an effective method for the further hydroxylation of other high value-added compounds.


Introduction
Cytochrome P450 enzymes (P450s, CYPs) are heme-containing enzymes that catalyze various types of chemical reactions on a variety of substrates (Hu et al., 2022b). Importantly, they are able to catalyze the regioselective and stereoselective oxidations of C-H bonds (Urlacher and Girhard, 2019). P450s are thought to be reliable, effective, and ecofriendly biocatalysts for the synthesis of valuable compounds in recombinant hosts. In addition, compared to the utilization of purified or extracted P450s, whole-cell biotransformation has shown a clear advantage by providing the necessary precursors, the expensive cofactors NAD(P)H, and suitable environments for catalytic reactions (Hu et al., 2022a). Moreover, to exploit the versatile P450s for industrial applications, Escherichia coli is a widely applied and efficient system for whole-cell biotransformation .
Flavonoids are one of the largest known groups of natural products, which are widely found in the plants (Havsteen, 2002). They have a phenyl benzopyrone structure (C6-C3-C6) and are mainly classified as flavones, flavanols, flavanones, flavanonols, and isoflavones ( Figure 1) (Middleton et al., 2000). They exhibit therapeutic and chemo-preventive effects on human health, including antioxidant activity (PG, 2000), antimicrobial activity (Cushnie and Lamb, 2005), anti-inflammatory activity (Pan et al., 2010), and anti-cancer properties (Ravishankar et al., 2013). In addition, they can be served as potential drug candidates to treat symptoms associated with the coronavirus disease (COVID-19) infection (Adhikari et al., 2021). However, the low water solubility and instability limit the pharmaceutical application of these flavonoid compounds (Chu et al., 2016). Hydroxylation is a common strategy to improve their solubility and stability (Lin and Yan, 2014). Moreover, the structural diversity and biological activity of flavonoids also can be improved through hydroxylation. For example, 7,3′,4′-trihydroxyisoflavone, the 3′-hydroxylated product of daidzein, exhibits better anti-cancer properties than daidzein and plays an essential role in suppressing ultraviolet B-induced skin cancer .
In this study, we report a bacterial whole-cell biocatalyst for the efficient hydroxylation of a variety of flavonoids. At first, the bacterial P450 sca-2 mut exhibiting outstanding 3′-hydroxylation activity towards flavonoids was selected from five P450s candidates. Then, the whole-cell activity of sca-2 mut towards flavonoids was enhanced by employing a new combination of redox partners and enzymatic engineering of sca-2 mut . Subsequently, the whole-cell activity was further enhanced by the optimization of whole-cell biocatalytic conditions. Finally, the whole-cell sca-2 mut biocatalyst was applied to efficiently produce eriodictyol, dihydroquercetin, luteolin, 7,3′,4′-trihydroxyisoflavone FIGURE 1 The skeleton structure of the main classes of flavonoids.

Plasmids and strains construction
All the plasmids, primers, and strains used in this study are listed in Supplementary Tables S1-S3, respectively.

Medium and culture conditions
Luria-Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and pH 7.0) was used for cloning and seeding cultures.

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To obtain seed cultures, colonies of the recombination strain grown from LB agar plates (2% agar, w/v) were inoculated into 50 mL test tubes containing 5 mL LB medium supplemented with 50 μg/mL kanamycin and incubated in a rotary shaker at 37°C and 220 rpm for 12 h. 1 mL of the seed cultures was transferred to 250 mL shaking flasks containing 50 mL Terrific Broth (TB) medium (12 g/L tryptone, 24 g/L yeast extract, 0.4% v/v glycerol, 0.017 M KH 2 PO 4 , and 0.072 M K 2 HPO 4 ) supplemented with 50 μg/mL kanamycin, 100 mg/L ALA and 20 mg/L FeSO 4 ·7H 2 O, and the cultures were then incubated at 37°C and 220 rpm. When the optical density at 600 nm (OD 600 ) reached 0.6-0.8, 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce enzyme expression. After induction, the cultures were incubated at 25°C for 20 h. For cultivation of HFLA-20 to HFLA-23 strains, 50 μg/mL kanamycin and 34 μg/mL chloramphenicol were added into the medium.

The hydroxylation of flavonoids by whole-cell biocatalysis
After cultivation, 50 mL of cells were harvested by centrifugation (8,000 rpm, 10 min), then washed twice with potassium phosphate buffer (100 mM, pH 8.0), and subsequently resuspended with 25 mL potassium phosphate buffer (100 mM, pH 8.0) containing 10% glycerol or 10% glucose. 25 mL of cell suspension (30 OD 600 ) was used for the whole-cell biocatalysis in 250 mL shaking flasks.
To examine the catalytic efficiency of hydroxylation of flavonoids, naringenin, dihydrokaempferol, kaempferol, apigenin, and daidzein (5 g/L in ethanol) was added to the cell suspension to give the final concentration of 100 mg/L, respectively. Whole-cell biocatalysis were performed at 30°C and 220 rpm for 12 h. Then, 1 mL of the whole-cell biocatalytic reaction solution was collected and extracted thrice with 1 mL ethyl acetate. The products were dried, dissolved in methanol, and subsequently analyzed using highperformance liquid chromatography (HPLC).

The optimal conditions for the hydroxylation of flavonoids by whole-cell biocatalysis
To optimize biocatalytic conditions, 25 mL of the cell suspension (30 of OD 600 ) was used for the bioconversion reaction in 250 mL shaking flasks. To investigate the effect of temperature on the catalytic activity, reactions were performed at pH 8.0 with the temperature ranging from 20°C to 40°C. To optimize pH, reactions were performed at 37°C and 220 rpm in potassium phosphate buffer (pH 6.0-8.0) or Tris-HCl buffer (pH 9.0).

FIGURE 2
Screening of the efficient bacterial P450s for the hydroxylation of flavonoids. (A) Schematic representation of the regioselective hydroxylation of naringenin by P450s with redox partners of CamA and CamB. (B) Schematic diagram of combining different P450s and redox partners to construct whole-cell biocatalyst plasmids. P450s included CYP105P2, CYP105D7, P450 moxA mut , CYP105A1 mut , and P450 sca-2 mut . (C) Titers of eriodictyol produced by whole-cell biocatalysts of different P450s using 100 mg/L naringenin. The data are shown as mean ± SD of three biological replicates.
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Analytical procedures
Cell growth was detected by measuring OD 600 using a spectrophotometer (UVmini-1240, Shimadzu Corporation, Japan).

Data analysis
All experiments were performed with three biological replicates. The data were analyzed by the software GraphPad Prism 8.0 and displayed as mean values ± standard deviation (SD) from triplicate experiments.
3 Results and discussion 3.1 Screening of the efficient bacterial P450s for the hydroxylation of flavonoids To screen the efficient bacterial P450s for the hydroxylation of flavonoids, CYP105D7 from S. avermitilis (Liu et al., 2016), sca-2 mut (CYP105A3) from S. carbophilus (Ba et al., 2013b), CYP105P2 from S. peucetius (Niraula et al., 2012), CYP105A1 mut from S. griseolus (Yasuda et al., 2017), and moxA mut (CYP105AB1) from N. recticatena (Kabumoto et al., 2009) were chosen as candidates to investigate the catalytic performance toward flavonoids. Since the CYP105 family is a three-component P450 enzyme, the most widely studied redox partners CamA (putidaredoxin reductase) and CamB (putidaredoxin) were employed in the whole-cell biocatalysis to transfer electrons from NAD(P)H to the heme-iron reactive center for O 2 activation. Thus, the genes encoding CamA and CamB were co-expressed with these five P450s genes using a pRSFDuet-1 plasmid in the C41(DE3) strain (Hu et al., 2022a), respectively, resulting in HFLA-1 to HFLA-5 strains. Subsequently, the hydroxylation of flavonoids by whole-cell as biocatalysts was compared using naringenin as a model substrate (Figures 2A, B).

Improving the catalytic activity of sca-2 mut by engineering redox partners
For the common bacterial three-component P450s, the process of transferring electron by redox partner is important for catalysis. However, the optimal redox partners for sca-2 mut are unknown (Zhang et al., 2018). To obtain the suitable redox partners for the hydroxylation of flavonoids by P450 sca-2 mut , the different combinations of flavodoxin and flavodoxin reductase were used to reconstitute the activity of P450 sca-2 mut , including the electron transfer proteins flavodoxin (Fld, FldA, or FldB) from E. coli in combination with the endogenous flavodoxin reductase Fpr (Bakkes et al., 2015), respectively, the flavodoxin (YkuN or YkuP) (Girhard et al., 2010) from B. subtilis in combination with E. coli Fpr, respectively, and the ferredoxin Fdx_1499 and ferredoxin reductase FdR_0978 from S. elongates PCC7942 (Sun et al., 2017) (Figure 3A). In addition, the chimeric protein was constructed by fusing the P450 sca-2 mut to the reductase domain of P450BM3 from B. megaterium ( Figure 3A).
The plasmids harboring the genes encoding different redox partners were transformed into the C41(DE3) strain, resulting in HFLA-6 to HFLA-12 strains. Subsequently, their performances in the hydroxylation of flavonoids were investigated using a final concentration of 100 mg/L naringenin as a substrate. Whole-cell biocatalysis was performed in potassium phosphate buffer containing glucose (1%, 2%, and 10% w/v) or glycerol (10% v/ v), respectively. The seven strains showed similar catalytic activity toward naringenin in the biocatalytic systems containing 1%, 2%, and 10% w/v of glucose (Supplementary Figure S1). The HFLA-7 strain (harboring plasmid pRSF-sca-2 mut -Fld-Fpr) had both the best catalytic performance towards naringenin in glycerol or glucose containing biocatalytic system, producing 38.6 ± 1.4 or 31.1 ± 1.0 mg/L eriodictyol, respectively ( Figure 3B). This result suggested that glycerol was more beneficial to the catalytic efficiency than glucose in the wholecell biocatalysis of HFLA-7 strain. Notably, the E. coli flavodoxin Fld was used in combination with Fpr for the first time to reconstitute the activity of P450s. In the recent study, FdR_ 0978/Fdx_1499 was the most promising redox partner for the Frontiers in Bioengineering and Biotechnology frontiersin.org in vitro activity of sca-2 towards mevastatin compared to the redox systems Adx/AdR and Pdx/PdR (Liu et al., 2022). However, the HFLA-6 strain (harboring plasmid pRSF-sca-2 mut -Fdx_1499-FdR_0978) produced 8.9 ± 0.2 and 9.7 ± 0.6 mg/L eriodictyol in the in vivo biocatalytic system containing glycerol or glucose, which were only 23.1% and 31.2% of the HFLA-7 strain, respectively ( Figure 3B). Therefore, the HFLA-7 strain was used for the following improvement.

Enhancing the catalytic activity of sca-2 mut by sequence-guided engineering
Based on the evolutionary information encapsulated in homologous protein sequences, the approach of consensus design has been employed to improve the stability (Porebski and Buckle, 2016) and activity (Yao et al., 2022) of proteins. Hence, the mutagenesis was designed to obtain the sca-2 variants by consensus design. Multiple sequence alignment was performed on the CYP105 family and the conserved arginine residues around the active site pocket were shown as red boxes ( Figure 4A). CYP105D7, sharing 53% identity of amino acid sequence with sca-2, has four arginine residues (Arg70, Arg81, Arg88, and Arg190) that form a wall of the substrate-binding pocket (Xu et al., 2015), and the double mutant R70A/R190A has a nearly 9-fold increase in the in vivo conversion rate of testosterone (Ma et al., 2019). The distal pocket of CYP105A1 contains three Arg residues (Arg73, Arg84, and Arg193) , and the double mutant R73A/ R84A exhibited a 319-fold higher K cat /K m for 25-hydroxylation towards the substrate 1α(OH) vitamin D 3 . Based on these simulated results, six residues of sca-2 mut (Arg77, Arg88, Arg93, Gly95, Ser 96, and Arg197) ( Figure 4B) were mutated, and the catalytic performance of the mutants on naringenin were examined.
Two single mutants, sca-2 mut R88A (HFLA-14 strain) and sca-2 mut S96A (HFLA-17 strain), exhibited 58% and 27% significantly increase in catalytic activity for naringenin compared to the HFLA-7 strain (harboring plasmid pRSF-sca-2 mut -Fld-Fpr), producing 60.9 ± 1.2 and 49.0 ± 2.9 mg/L eriodictyol using 100 mg/L naringenin as a substrate, respectively ( Figure 4C). Then, the double mutant sca-2 mut R88A/S96A (HFLA-19 strain) was constructed, and the catalytic activity was further increased by 10% compared to the best single mutant sca-2 mut R88A (HFLA-14 strain), producing 67.2 ± 1.7 mg/L eriodictyol. To analyze the reason for the enhanced yield, the homology models for the sca-2 mut and mutant sca-2 mut R88A/S96A were constructed and checked (Supplementary Figure S2). Subsequently, the analysis of molecular docking was performed using the CDOCKER tool of DS 2019 with substrate naringenin as the ligand (Figures 4D, E). Although the amino acids Arg 88 and Ser 96 did not directly interact with the substrate naringenin, the hydrogen bond around the substrate increased in the sca-2 mut R88A/S96A model compared to the sca-2 mut model (Figures 4D, E; Supplementary Figure S3). In addition, the increased hydrophobic interaction of Ala 88 and Ala 96 with surrounding amino acids may lead to a more flexible of substrate access (Supplementary Figure S4).

Improving the efficiency of electron transfer by introducing different scaffolds
The efficient electron transfer between P450s and redox partners is important for the biosynthesis of natural products (Park et al., 2022a). Therefore, DNA scaffolds , Protein scaffolds (Dueber et al., 2009), Photorhabdus luminescens CipA scaffold (Wang et al., 2017), and P. luminescens CipB scaffold (Wang et al., 2017) were applied to assemble Fpr, Fld, and sca-2 mut R88A/S96A. Different scaffolds were fused to the N terminus or C terminus of these three enzymes and their effects on the catalytic performance of naringenin were investigated. At first, DNA scaffolds, protein scaffolds, CipA scaffold, and CipB scaffold were respectively fused to the N terminus of Fpr, Fld, and sca-2 mut R88A/S96A in the HFLA-19 strain to generate HFLA-20, HFLA-22, HFLA-24, and HFLA-26 strains ( Figure 5A). The titers of eriodictyol in these four strains decreased by 33.7%-61.7% compared to the HFLA-19 strain, indicating that the scaffolds fused to the N terminus of the three enzymes resulted in an overall decrease in whole-cell activity ( Figure 5B).
Then, the scaffolds were fused to the C terminus of Fpr, Fld, and sca-2 mut R88A/S96A in the HFLA-19 strain, respectively, obtaining the HFLA-21, HFLA-23, HFLA-25, and HFLA-27 strains. These four strains produced 100.1%, 94.8%, 65.3%, and 53.7% of the eriodictyol titers of the HFLA-19 strain, respectively, indicating that the use of scaffolds to assemble of P450s and redox partners did not further improve the titers of eriodictyol ( Figure 5B). In the previous reports, these scaffolds were used to assemble enzymes to enhance the synthesis of natural products in growing cells (Dueber et al., 2009;Xu et al., 2020;Park et al., 2022a). During the fermentation of growing cells, products are synthesized from growth substrates by the natural metabolism of the host cells and are accompanied in the fermentation broth by metabolic intermediates that make downstream processing complicated (Ladkau et al., 2014;Lee and Kim, 2015). In biotransformation, the cell growth and production phase are separated, and the use of resting cells can convert substrates to desired products (Lin and Tao, 2017). In addition, the use of resting cells is a good alternative when the optimal pH, temperature, or medium composition for biotransformation differs from the values that allow optimal growth conditions (de Carvalho, 2017). Thus, the scaffolds were not suitable for the whole-cell catalysis with P450 sca-2 mut R88A/S96A using resting cells.  Frontiers in Bioengineering and Biotechnology frontiersin.org

The optimization of whole-cell biocatalytic conditions for the HFLA-19 strain
To obtain the optimal conditions of whole-cell biocatalysis, the suitable temperature and pH were optimized using the HFLA-19 biocatalyst. At first, the effect of biocatalytic temperature ranging from 20°C to 40°C on the production of eriodictyol was examined using 100 mg/L naringenin as a substrate. The results showed that the titer of eriodictyol increased with the increase of biocatalytic temperature ( Figure 6A). 71.3 ± 0.7 mg/L of eriodictyol was produced at the biocatalytic temperature at 37°C, and the titer did not increase when the biocatalytic temperature was higher than 37°C. Subsequently, the effect of pH values ranging from 6.0 to 9.0 on the production of eriodictyol was examined, and the highest titer of eriodictyol (71.3 ± 0.7 mg/L) was obtained at pH 8.0°C and 37°C ( Figure 6B).
Since P450s are heme-containing enzymes, increasing the intracellular supply of heme can enhance the overall activity of whole-cell biocatalysts . However, the direct addition of different final concentrations of heme (5, 10, 20, 30, and 40 mg/L) to the medium did not significantly increase the titer of eriodictyol due to the weak import of heme in the C41(DE3) strain (Supplementary Figure S5), which is consistent with the previous study (Zhao et al., 2022). Since the uptake of the heme precursor (5aminolevulinic acid, ALA) was efficient in E. coli (Verkamp et al., 1993), the effect of adding different final concentrations of ALA (50, 100, 200, 300, and 400 mg/L) on biocatalysis was investigated. The highest titer of eriodictyol could reach 74.3 ± 0.4 mg/L when 100 mg/L ALA was added to the medium ( Figure 6C). In addition, the supplementation with iron also helps in the synthesis of heme. Based on adding 100 mg/L ALA in the medium, different final concentrations of FeSO 4 ·7H 2 O (5, 10, 20, 30, and 40 mg/L) were added into the medium, respectively, and 77.3 ± 0.6 mg/L eriodictyol was produced when 20 mg/L FeSO 4 ·7H 2 O was supplied ( Figure 6D). Hence, the 72.5% of molar conversion rate is higher than the highest molar conversion rate (59.3%) reported so far for the heterologous expression of the F3′H from Gentiana triflora and cytochrome P450 reductase from Arabidopsis thaliana in engineering E. coli (Table 1) (Liu et al., 2022).

Conclusion
In this study, an efficient bacterial whole-cell P450 biocatalyst was obtained by mining the suitable P450 enzymes, engineering redox partners, protein engineering, and the optimization of wholecell biocatalytic conditions. By using the sca-2 mut R88A/S96A wholecell biocatalyst, eriodictyol, dihydroquercetin, quercetin, luteolin, and 7,3′,4′-trihydroxyisoflavone were produced with the titers of 77.3, 66.3, 5.7, 31.8, and 75.1 mg/L, respectively, in a reaction system containing a final concentration of 100 mg/L substrate. To the best of our knowledge, this is the first report of C-3′ hydroxylation of flavonoids by P450 sca-2 (CYP105A3), expanding the substrate spectrum of sca-2. In addition, the conversion rates of eriodictyol, dihydroquercetin, luteolin, and 7,3′,4′trihydroxyisoflavone were the highest conversion rates obtained so far by whole-cell biocatalysis of P450s. This study demonstrates a versatile P450 whole-cell biocatalyst for the efficient hydroxylation of flavonoids, providing a potential biocatalyst for application in synthetic biology.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.